The dream of watching a protein function in real time with near atomic resolution has been realized using picosecond time-resolved Laue crystallography, an experimental methodology first developed by the Anfinrud group at the ESRF in Grenoble, France. To advance this capability further, we have launched a major effort to pursue picosecond time-resolved X-ray science at the Advanced Photon Source (APS) in Argonne, IL. In addition to our efforts to develop the infrastructure required to make these measurements, which is summarized in a separate report, we continue to develop TReX, an in-house software package designed to analyze time-resolved Laue data. This effort has proven to be much more demanding than envisioned at the outset, but represents a critical component in our research. We are currently overcoming remaining issues and are working on a major update to this software package: TReX-II. The first step in analyzing crystal diffraction data, whether acquired with monochromatic or polychromatic X-ray radiation, is the indexing of diffraction spots recorded on a two-dimensional detector. Robust auto-indexing algorithms have long existed for monochromatic diffraction images, but Laue diffraction images, which are generated with a polychromatic X-ray source, are not amenable to those methods. Consequently, the analysis of Laue diffraction data has generally proven to be a time-consuming, off-line process that has required a significant amount of face time in front of a computer. To address this problem, a robust zone-based algorithm for auto-indexing Laue diffraction images was developed by Dr. Eric Henry. When the image center and distance between the sample and the detector are prescribed, spots on the detector plane can be mapped onto a locus of possible zone-vector directions, the consensus of which identifies zone-vectors suitable for determining the orientation of the crystal. As an added benefit, zone-vector consensus provides a criterion for optimizing the center of the detector, knowledge of which is crucial to accurately predict the Laue diffraction pattern. Once the Laue pattern is predicted, the spot intensities need to be integrated, scaled, and merged with results from numerous crystal orientations. Finally, the merged results are Fourier transformed to generate time-resolved electron density maps. The integration methods employed by our group thus far are derivative of PROW, a software package developed by Dr. Dominique Bourgeois for PRofile integration of Overlapping and Weak spots. We have recently discovered that the precision and accuracy of the integration suffers from numerous systematic errors;these errors contribute much noise to the diffraction data, and adversely affect the quality of electron density maps constructed from those data. These problems came to light when analyzing data recently acquired using a novel protocol that allowed us to acquire an X-ray diffraction time series involving 37 images at a single orientation from a single crystal. To minimize the extent of radiation damage, which would destroy the protein crystal if that many images were acquired from a single spot, we translated the crystal stepwise along its length between single X-ray pulses, thereby distributing the radiation damage across the entire length of the crystal. This protocol requires long crystals, and was tested using photoactive yellow protein crystals 0.6-mm in length. Examination of the spot intensities across the extensive time series unveiled numerous sources of systematic errors that compromise the accuracy of the integration methods currently employed by TReX. We are in the process of breaking away from PROW-based methods and are developing new integration and scaling methods that avoid the pitfalls that have plagued this and other Laue processing software packages. Key to this success is our ability to acquire many images at a common orientation, which provides sufficient statistics for accurate scaling information and to identify outliers in the time series. Moreover, we have discovered that the MarCCD X-ray detector suffers from dramatic pixel-to-pixel variations in the statistical uncertainty, an effect which came to light when characterizing time-resolved SAXS/WAXS scattering patterns. Each active pixel in this 2048x2048 detector has now been characterized in detail and that information will soon be used to improve the integration accuracy, refine estimates of the integration precision, and enhance the statistical sensitivity for outlier rejection and correction. Once diffraction spots are integrated accurately, redundant observations from other orientations must be merged to assemble a data set that is as complete as possible. Merging redundant Laue data requires both image scaling and wavelength normalization, each of which is error prone. Thus, the quality of Laue data has traditionally been inferior to data acquired with monochromatic methods. We are currently working on a ratio method that eliminates the need to scale redundant observations acquired at different orientations, as scaling is automatically accomplished by taking the ratio of laser on and laser off observations acquired in the time series. We anticipate that electron density maps computed according to merged ratios will provide much greater structural detail than achieved in the past. When the level of photoactivation is small, as is often the case, the electron density maps recovered are dominated by the ground or starting equilibrium state. To characterize the structures of short-lived intermediates, we first subtract the contribution from the ground state and rescale the remaining electron density to 100%. This procedure degrades the signal-to-noise (S/N) ratio by the scale factor used in the extrapolation, and makes interpretation of the extrapolated electron density maps even more challenging. A global analysis method developed by our group for processing time-resolved spectroscopic data has recently been extended to time-resolved electron density data. This method allows us to recover by non-linear least-squares methods the time-independent electron density maps for each state in a kinetic model. The S/N ratio for these maps are enhanced relative to the individual snapshots in accordance with the number of time frames in which that structure is represented. Moreover, this approach allows us to refine rate coefficients for structural change. This methodology was first tested on a recently acquired PYP data set, and the results obtained are very promising. Indeed, the lifetime of an unusually-twisted structural intermediate (0.8 ns) seems to be significantly shorter than the 3 ns lifetime reported earlier via prior time-resolved optical spectroscopy. Note that the optical absorption spectrum of a chromophore is only indirectly influenced by protein structural changes, whereas the structural changes observed by time-resolved Laue crystallography are determined directly. More work is needed to assess the accuracy of the lifetime recovered by time-resolved crystallography.